Antitumor agents albacarcins V and M

ABSTRACT

Novel antitumor antibiotics designated herein as albacarcins M and V are produced by fermentation of Streptomyces albaduncas strain C-38291 (ATCC 39151). The new antibiotics possess antibacterial activity and also inhibit the growth of mammalian tumors such as P388 leukemia in mice.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to two novel polycyclic antitumorantibiotic compounds designated herein as albacarcin M and albacarcin Vand to their preparation by fermentation of a new strain of Streptomycesalbaduncus designated herein as Streptomyces albaduncus C38291 (ATCC39151).

2. Description of the Prior Art

Isolation of the polycyclic antitumor agents named gilvocarcins V, M andE from the fermentation broth of Streptomyces anandii subsp. araffinosusstrain C-22437 (ATCC 31431) is disclosed in J. Antibiotics 34: 1544-1555(1981). The gilvocarcin antibiotics have the structures shown below:##STR1##

As noted in the above-mentioned J. Antibiotics reference, gilvocarcins Vand M were previously reported in J. Antibiotics 34: 266-275 (1981) asbeing isolated from the fermentation broth of Streptomyces gilvotanareus(NRRL 11382).

Gilvocarcin V and gilvocarcin M are the same as toromycin A andtoromycin B, respectively, disclosed in Agric. Biol. Chem. 44: 1157-1163(1980).

The antitumor antibiotic designated as ravidomycin Ia is disclosed inCan. J. Chem. 59: 3018-3020 (1980). Ravidomycin Ia, isolated from thefermentation broth of Streptomyces ravidus, has the structure ##STR2##Ravidomycin Ia thus possesses the same aglycone moiety as gilvocarcin V(toromycin A).

The antitumor antibiotic designated as virenomycin is disclosed inAntibiotiki 22: 963-967 (1977); see also Antibiotiki 23: 675-676 (1978),Soviet Journal of Bioorganic Chemistry 4(8): 798-802 (1978) andEncyclopedia of Antibiotics, Second Edition, J. S. Glasby, Ed., pg. 457,Wiley-Interscience, Chichester-New York-Brisbane-Toronto, 1979,Virenomycin is isolated from the fermentation broth of Streptomycesvirens strains 3831 and 3931/183. While the sugar moiety has beenidentified (see Soviet Journal of Bioorganic Chem. reference above), thestructure of the remaining portion has never been reported. Virenomycin,which has the same sugar moiety as the antibiotics of the presentinvention, is reported to have a "comparatively low antitumor activityand narrow spectrum" (Antibiotiki 22: 963-967 at 967, 1977).

SUMMARY OF THE INVENTION

The present invention provides the polycyclic antitumor antibioticsalbacarcin M and albacarcin V and a process for their preparation andisolation in a purified state substantially free of co-producedsubstances. The new antibiotics are obtained by cultivating analbacarcin M- and/or albacarcin V-producing strain of Streptomycesalbaduncus having the identifying characteristics of strain C38291 (ATCC39151) in an aqueous nutrient medium containing assimilable sources ofcarbon and nitrogen under submerged aerobic conditions until asubstantial amount of albacarcin M and albacarcin V are produced by saidorganism in said culture medium and then recovering the albacarcin M andalbacarcin V from said culture medium in substantially pure form.

The albacarcin M and albacarcin V antibiotics of the present inventionhave been found to exhibit antimicrobial activity and to inhibit thegrowth of tumors in experimental animals.

DETAILED DESCRIPTION

The albacarcin M and albacarcin V antibiotics provided by the presentinvention have been determined to have the structures ##STR3## wherein Ris --CH₃ (albacarcin M) or --CH═CH₂ (albacarcin V).

Compound albacarcin V is a yellow amorphous solid having a molecularformula of C₂₈ H₂₈ O₉ and a molecular weight of 508.555. It is composedof the elements carbon, hydrogen and oxygen. Elemental analysis data isas follows:

Calculated for C₂₈ H₂₈ O₉.H₂ O: C, 63.87; H, 5.74; O(by difference),30.39.

Found: C, 63.54; H, 5.58; O(by difference), 30.88.

The infrared absorption spectrum of albacarcin V when pelleted in KBrexhibits characteristic bands at the following frequences expressed inreciprocal centimeters: 3400, 2980, 2940, 1705, 1625, 1610, 1590, 1510,1490, 1450, 1435, 1370, 1335, 1330, 1250, 1190, 1160, 1150, 1130, 1065,1010, 995, 975, 945, 915, 870, 850, 830, 780 and 720.

The ultraviolet absorption spectrum of albacarcin V was determined inmethanol (0.02096 g/l). Observed absorption maxima and absorptivitieswere as follows: 392 nm (ε=7272), 286 nm (ε=33,970), 246 nm (ε=33,259).

Optical rotation values for albacarcin V are as follows:

    [α].sub.Na(589).sup.24° (c=0.3, DMSO)=-9.7°

    [α].sub.Hg(578).sup.24° (c=0.3, DMSO)=-10°

    [α].sub.Hg(546).sup.24° (c=0.3 DMSO)=-14°

A proton magnetic resonance spectrum of albacarcin V dissolved in DMSOwas determined with a Bruker Model WM-360 Spectrometer operating at 360MHz. Observed chemical shifts and pattern descriptions are as follows:δ9.82 (s, 1H, ArOH), 8.48 (s, 1H, H-11), 800 (s, 1H, H-7), 7.85 (d,J=8.22, 1H, H-3), 7.74 (s, 1H, H-9), 6.98 (d J=8.22, 1H, H-2), 6.93 (dd,J=15.82 and 10.76, 1H, --CH═CH₂), 6.14 (d, J=15.82, 1H, --CH═CH--), 6.04(d, J=7.59, 1H, H=1'), 5.51 (d, J=10.76, 1H, --CH═CH--, 4.60 (d, J=6.32,1H, OH), 4.52 (m, 1H, H-5'), 4.17 (s, 3H, OCH₃), 4.12 (s, 3H, OCH₃),3.69 (t, J=9.43, H-4'), 3.16 (d, J=6.29, H-3'), 1.21 (s, 3H, 3'--CH₃),1.03 (d, J=6.29, 3H, 5'--CH₃)

A carbon--13 magnetic resonance spectrum of albacarcin V dissolved inDMSO was determined with a Bruker Model WM-360 Spectrometer operating at90.5 MHz. Observed chemical shifts and assignments are as follows:

    ______________________________________                                        No.    Chemical Shift (ppm)                                                                         Multiplicity                                                                              Assignment                                  ______________________________________                                         1     159.8          s           6                                            2     157.3          s           10                                           3     153.2          s           1                                            4     151.8          s           12                                           5     142.4          s           4b                                           6     138.7          s           8                                            7     135.1          d           8a                                           8     129.3          d           3                                            9     128.0          s           4                                           10     125.1          s           4a                                          11     122.9          s           6a                                          12     121.9          s           10a                                         13     118.9          d           9                                           14     117.1          t           8b                                          15     115.1          s           12a                                         16     114.6          d           2                                           17     113.2          s           10b                                         18     112.1          d           7                                           19     101.5          d           11                                          20     75.8           d           1'                                          21     74.6           d           5'                                          22     73.1           s           3'                                          23     72.6           d           4'                                          24     70.7           d           2'                                          25     56.6           q           OCH.sub.3                                   26     56.2           q           OCH.sub.3                                   27     23.8           q           3'-CH.sub.3                                 28     17.0           q           6'                                          ______________________________________                                    

Compound albacarcin M is a yellow amorphous solid having a molecularformula of C₂₇ H₂₈ O₉ and a molecular weight of 496.484.

The infrared absorption spectrum of albacarcin M when pelleted in KBrexhibits characteristic bands at the following frequencies expressed inreciprocal centimeters: 3400, 2980, 2940, 1710, 1615, 1590, 1505, 1490,1450, 1430, 1370, 1340, 1300, 1249, 1190, 1165, 1145, 1130, 1060, 1030,1010, 990, 975, 955, 850, 820, 780, 715, 650, 615.

The ultraviolet absorption spectrum of albacarcin M was determined inmethanol (0.01019 g/l). Observed absorption maxima and absorptivitieswere are follows: 382 nm (ε=9731) 305 nm (ε=10,724)275 nm (ε=31,825),266 nm (ε=24,973) and 244 nm (ε=43,343).

Optical rotation values for albacarcin M are as follows:

    [α].sub.Na(589.3).sup.24° (c=0.03, DMSO)=-14.9°

    [α].sub.Hg(578).sup.24° (c=0.03, DMSO)=-14.9°

    [α].sub.Hg(546).sup.24° (c=0.3, DMSO)=-22.3°

A proton magnetic resonance spectrum of albacarcin M dissolved in DMSOwas determined with a Bruker Model WM-360 Spectrometer operating at 360MHz. Observed chemical shifts and pattern descriptions are as follows:δ9.83 (s, 1H, ArOH), 8.47 (s, 1H, H-11), 7.84 (d, J=9.49, 1H, H-3), 7.77(s, 1H, H-7) 7.49 (s, 1H, H-9), 6.96 (d, J=9.49, 1H, H-2), 6.04 (d,J=9.49, 1H, H-1'), 4.59 (d, J=7.91, 1H, H-5'), 4.53 (m, 1H, OH), 4.20(s, 1H, OH), 4.18 (d, J=9.49, 1H, OH), 4.11 (s, 6H, OCH₃), 3.70 (t,J=9.49, 1H, H-4'), 3.16 (d, J=9.49, 1H, H-3'), 1.27 (s, 3H, H-8a), 1.02(d, J=7.91, 3H, H-6').

A carbon--13 magnetic resonance spectrum of albacarcin M dissolved inDMSO was determined with a Bruker Model WM-360 Spectrometer operating at90.5 MHz. Observed chemical shifts and assignments are as follows:

    ______________________________________                                        No.    Chemical Shift (ppm)                                                                         Multiplicity                                                                              Assignment                                  ______________________________________                                         1     159.9          s           6                                            2     156.8          s           10                                           3     153.2          s           1                                            4     151.6          s           12                                           5     141.9          s           4b                                           6     140.3          s           8                                            7     129.1          d           3                                            8     127.8          s           4                                            9     125.1          s           4a                                          10     121.4          s           6a                                          11     121.0          d           9                                           12     120.8          s           10a                                         13     118.7          d           2                                           14     114.9          s           12a                                         15     113.3          s           10b                                         16     111.7          d           7                                           17     101.5          d           11                                          18     75.8           d           1'                                          19     74.6           d           5'                                          20     73.1           s           3'                                          21     72.6           d           4'                                          22     70.7           d           2'                                          23     56.3           q           10-OCH.sub.3                                24     56.1           q           12-OCH.sub.3                                25     23.8           q           3'-CH.sub.3                                 26     21.0           q           8-CH.sub.3                                  27     17.0           q           6'                                          ______________________________________                                    

The antibiotics of the present invention may be prepared according tothe fermentatiion and isolation procedures described below.

The Producing Organism

The antibiotics of the present invention may be prepared by cultivatingan albacarcin M and/or albacarcin V-producing strain of Streptomycesalbaduncus in an aqueous nutrient medium under submerged aerobicconditions until a substantial amount of albacarcin M and/or albacarcinV is produced by said organism in said culture medium.

The preferred producing organism is a novel strain of Streptomycesalbaduncus designated herein as Streptomyces albaduncus strain C38291.This strain was isolated from a soil sample collected in Columbia. Abiologically pure culture of strain C38291 has been deposited with theAmerican Type Culture Collection, Rockville, Md., and added to theirpermanent collection of microorganisms as ATCC 39151.

Characteristics of the above-mentioned preferred strain are described indetail below.

Strain C38291 (ATCC 39151)

Strain C38291 forms substrate and aerial mycelia (0.5μ in width). Bothmycelia are long, well branched and not fragmented into short filaments.Spore chains are born monopodially on aerial mycelia or at hyphal tips.The spore chains form open or irregular short spirals and hooks, andcontain 10-30 spores in a chain. The spores are spherical, oval orbarrel-shaped (0.8 by 0.8-0.9μ ), and have a spiny surface. Sporangia,motile spores and sclerotia are not observed.

Strain C38291 grows well in all media including Czapek's agar. Theaerial mycelium is poorly formed in most media such as Czapek's and ISP(International Streptomyces Project) agars, but forms abundantly onBennett's agar. Gray aerial mycelium is formed in ISP medium Nos. 3 and7, and Bennett's agar. Yellow or grayish olive green aerial mycelia areobserved in Czapek's agar, ISP medium Nos. 2, 4 and 5, andglucose-asparagine agar. Therefore, the aerial mycelium of strain C38291is similar to that of three color series, Yellow, Green and Gray. Yellowand green colors of the aerial mycelium of strain C38291 are likely tobe derived from the diffused yellow or dark olive green pigments of thesubstrate mycelium. Thus, the intrinsic color of the aerial mycelium ofstrain C38291 is deduced to be gray. Strain C38291 produces reddishdiffusible pigment in ISP medium Nos. 1, 2 and 7 and Bennett's agar, thecolor being red in alkali and yellow in acid. Melanoid pigment is notproduced. The cultural characteristics are shown below in Table I.

                  TABLE I                                                         ______________________________________                                        Cultural Characteristics of Strain C38291                                     ______________________________________                                        Tryptone-yeast extract broth                                                                  G:    moderate; forms light                                   (ISP No. 1)           olive brown pellets                                                     D:    yellowish pink (26)                                     Sucrose-nitrate agar                                                                          G:    moderate                                                (Czapek's agar) R:    dark yellowish brown (78)                                               A:    poor, light grayish olive                                                     (109)                                                                   D:    deep yellowish brown (75)                               Glucose-asparagine agar                                                                       G:    moderate                                                                R:    colorless to dark grayish                                                     yellow (91)                                                             A:    scant to poor, yellowish                                                      gray (93) to yellowish                                                        white (92)                                                              D:    none                                                    Glycerol-asparagine agar                                                                      G:    moderate                                                                R:    dark yellowish brown (78)                                               A:    poor, yellowish gray (93)                                                     to light gray (264)                                                     D:    none to deep yellowish                                                        brown (75)                                              Inorganic salts-starch agar                                                                   G:    moderate                                                (ISP No. 4)     R:    light olive brown (94)                                                  A:    moderate, light gray (264)                                              D:    none to deep yellowish                                                        brown (75)                                              Tyrosine agar   G:    moderate                                                (ISP No. 7)     R:    deep yellowish brown (75)                                                     to dark brown (59)                                                      A:    poor to moderate,                                                             yellowish gray (93) to                                                        light gray (264)                                                        D:    dark brown (59)                                         Nutrient agar   G:    poor                                                                    R:    colorless                                                               A:    scant, yellowish white                                                        (92)                                                                    D:    none                                                    Yeast extract-malt extract                                                                    G:    moderate                                                agar (ISP No. 2)                                                                              R:    light yellowish brown (76)                                                    to dark olive (108)                                                     A:    poor to moderate, light                                                       olive gray (112) to                                                           medium gray (265)                                                       D:    deep reddish brown (41)                                 Oatmeal agar    G:    poor                                                    (ISP No. 3)     R:    colorless to grayish                                                          yellow (90)                                                             A:    poor, light olive gray                                                        (112) to medium gray (265)                                              D:    none to light olive brown                                                     (94)                                                    Bennett's agar  G:    moderate                                                                R:    dark olive brown (96) to                                                      dark reddish brown (44)                                                 A:    moderate, grayish yellowish                                                   brown (80)                                                              D:    medium reddish brown (43)                               Peptone-yeast extract-iron                                                                    G:    poor                                                    agar (ISP No. 6)                                                                              R:    colorless                                                               A:    none                                                                    D:    dark olive brown (96)                                   ______________________________________                                         *observed after incubation at 28° C. for 3 weeks                       **Abbreviation: G = Growth; R = Reverse color; A = Aerial mycelium; D =       Diffusible pigment                                                            ***Color and number in parenthesis follow the color standard in "Kelly        K.L. & D.B. Judd: ISCCNBS colorname charts illustrated with Centroid          Colors. U S Dept. of Comm. Cir. 553, Washington, D.C., Nov., 1975".      

The growth of strain C38291 is inhibited by streptomycin ortetracycline, but not by benzylpenicillin, ampicillin or erythromycin.The sodium chloride tolerance is seen at 8% or less, but not at 10% ormore. Melanin is not produced from L-3,4-dihydroxy phenylalanine(L-DOPA). Among eleven diagnostic sugars described in Bergey's Manual,8th ed. (1974), only raffinose is not utilized by strain C38291. Thephysiological characteristics and utilization of carbohydrates are shownin Tables II and III, respectively.

                  TABLE II                                                        ______________________________________                                        Physiological Characteristics of                                              Strain C38291                                                                 Test       Response      Method or medium used                                ______________________________________                                        Range of temp-                                                                           Maximal growth at                                                                           Bennett's agar                                       erature for                                                                              37° C. to 43° C.                                     growth     Moderate growth at                                                            28° C. and 45° C. No                                            growth at 7° C. and                                                    50° C.                                                      Gelatin    Liquefied     Glucose-peptone-gelatin                              liquefaction             medium                                               Starch     Hydrolyzed    Starch agar plate                                    hydrolysis                                                                    Reactions in                                                                             Not coagulated and                                                                          Difco skimmed milk                                   skimmed milk                                                                             completely                                                                    peptonized                                                         Formation of mel-                                                                        Not produced  Tyrosine agar, peptone-                              anoid pigment            yeast-iron agar and                                                           tryptone-yeast extract                                                        broth                                                Tyrosinase re-                                                                           Negative      Arai's method*                                       action                                                                        Nitrate reduction                                                                        Negative      Czapek's sucrose-nitrate                                                      broth and glucose-                                                            yeast extract broth                                  Acid tolerance                                                                           Growth at pH 4.5.                                                                           Yeast extract-malt                                              No growth at  extract agar                                                    pH 4.0                                                             NaCl tolerance                                                                           Growth at 8% or                                                                             1% yeast extract, 2%                                            less. No growth at 10%                                                                      soluble starch, 1.5%                                            or more.      agar                                                 ______________________________________                                         *Arai, T. and Y. Mikami: Chromogenicity of Streptomyces. Appl. Microbiol.     23: 402-406, 1972.                                                       

                  TABLE III                                                       ______________________________________                                        Carbohydrate Utilization of                                                   Strain C38291                                                                 Glycerol          +                                                           D(-)-Arabinose    ±                                                        L(+)-Arabinose    +                                                           D-Xylose          ±                                                        D-Ribose          +                                                           L-Rhamnose        +                                                           D-Glucose         +                                                           D-Galactose       +                                                           D-Fructose        +                                                           D-Mannose         +                                                           L(-)-Sorbose      -                                                           Sucrose           +                                                           Lactose           +                                                           Cellobiose        +                                                           Melibiose         -                                                           Trehalose         +                                                           Raffinose         -                                                           D(+)-Melezitose   -                                                           Soluble starch    +                                                           Cellulose         -                                                           Dulcitol          -                                                           Inositol          ±                                                        D-Mannitol        +                                                           D-Sorbitol        -                                                           Salicin           +                                                           ______________________________________                                         observed after incubation at 28° C. for 3 weeks.                       Basal medium: PridhamGottlieb's inorganic medium                              Abbreviation: +: positive utilization, -: negative utilaization, ±:        doubtful utilization                                                     

The morphological, cultural and physiological characteristics of strainC38291 described above are consistent with those of the genusStreptomyces. According to the descriptions of Bergey's Manual, strainC38291 should be placed in the following species group: section Spiralesor Retinaculum-Apertum, gray series, non-chromogenic and spiny sporesurface. This group includes 24 species. Based on the descriptions ofISP species, strain C38291 resembles Streptomyces canus ISP 5017, S.albaduncus strain No. 13246-28-4-27 (J. Antibiotics 17: 39-47 (1964),and S. olivoviridis ATCC 15882 in its spore chain morphology, NaCltolerance and carbohydrate utilization. Strain C38291 is similar to S.griseoincarnatus ATCC 23917 in the carbohydrate utilization pattern andthe formation of reddish diffusible pigment, but differs distinctly fromthe latter in its grayish aerial mycelium. As a result of directcomparison with the above-mentioned four reference Streptomyces species,strain C38291 is considered to be most similar to strain No. 13246-28-4-27 of Streptomyces albaduncus ATCC 14698, although some differences arenoted between the two strains such as the formation of non-diffusiblegreen pigment. Strain C38291, therefore, is considered to be a novelstrain of Streptomyces albaduncus.

Since the Streptomyces are easily mutated naturally or artificially, thepresent invention includes within its scope Streptomyces albaduncusstrain C38291 (ATCC 39151) and all natural and artificial albacarcin M-and/or albacarcin V-producing variants and mutants thereof.

Antibiotic Production

The albacarcin V and M antibiotics of the present invention are preparedby cultivating an albacarcin V- and albacarcin M-producing strain ofStreptomyces albaduncus, preferably a strain of Streptomyces albaduncushaving the identifying characteristics of strain C38291 (ATCC 31951) ora mutant or variant thereof, in a conventional aqueous nutrient medium.The organism is grown in a nutrient medium containing known nutritionalsources for actinomycetes, i.e. assimilable sources of carbon andnitrogen plus optional inorganic salts and other known growth factors.Submerged aerobic conditions are preferably employed for the productionof large quantities of antibiotic, although for production of limitedamounts, surface cultures and bottles may also be used. The generalprocedures used for the cultivation of other actinomycetes areapplicable to the present invention.

The nutrient medium should contain an appropriate assimilable carbonsource such as glycerol, L(+)-arabinose, ribose, glucose, fructose,mannose, sucrose, lactose, cellobiose, soluble starch or mannitol. Asnitrogen sources, ammonium chloride, ammonium sulfate, urea, ammoniumnitrate, sodium nitrate, etc. may be used either alone or in combinationwith organic nitrogen sources such as peptone, meat extract, yeastextract, corn steep liquor, soybean powder, cotton seed flour, etc. Theymay also be added if necessary nutrient inorganic salts to providesources of sodium, potassium, calcium, ammonium, phosphate, sulfate,chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron,and the like.

Production of the albacarcin V and M antibiotics can be effected at anytemperature conducive to satisfactory growth of the producing organism,e.g. 25°-45° C., and is most conveniently carried out at a temperatureof around 27°-32° C. Ordinarily, optimum production is obtained inshaker flasks after incubation periods of 6-8 days. When tankfermentation is to be carried out, it is desirable to produce avegetative inoculum in a nutrient broth by inoculating the broth culturewith a slant or soil culture or a lyophilized culture of the organism.After obtaining an active inoculum in this manner, it is transferredaseptically to the fermentation tank medium. Antibiotic production maybe monitored by high performance liquid chromatography assay or by aconventional biological assay.

Isolation and Purification

A preferred isolation and purification scheme for albacarcins V and M isshown below. ##STR4##

To elaborate on the isolation and purification procedure, albacarcins Vand M exist mainly mycelial bound, although a small portion of theantibiotic activity is in the liquid part of the fermented broth. Thewhole fermentation broth is first extracted with a suitablewater-immiscible organic solvent such as ethyl acetate. Filter aid maybe optionally employed at this stage to facilitate filtration. Theorganic solvent extract is then filtered. Filtrate is then concentratedand filtered. The mycelial mat from the harvested broth is extractedwith the organic extraction solvent, e.g. ethyl acetate, and the organicsolvent extract filtered. Filtrate from the mycelial extraction step iscombined with filtrate from the broth extraction. The combined filtratesare then concentrated and the active solids precipitated with a suitableantisolvent such as heptane or Skellysolve B. The precipitated solidsare collected by filtration and subjected to a liquid partitionprocedure.

For the liquid partition, the crude precipitated solids are suspended ina mixture of methanol and Skellysolve B. The resultant suspension istransferred to a separatory funnel and diluted with water. Upon shakingand separation of the phases, the aqueous methanol phase is transferredto a second separatory funnel, diluted with water and extracted withcarbon tetrachloride which has been previously saturated with 25%aqueous methanol. The aqueous methanol phase is separated, diluted withwater and extracted with chloroform which has previously been saturatedwith 35% aqueous methanol. All of the suspended solids dissolve at thispoint. The chloroform extract is then concentrated to dryness to providea crude complex of albacarcins V and M.

The crude antibiotic complex is then subjected to silica gel columnchromatography. Elution is carried out with a linear gradient ofmethylene chloride to 6% absolute ethanol in methylene chloride. Activefractions are pooled and concentrated in vacuo to provide a mixture ofalbacarcin V and M.

Final separation and purification of the albacarcin V and M componentsmay be achieved by preparative high performance liquid chromatography asdescribed below in Example 2.

Biological Properties of Albacarcin V and M

The antibacterial activities of albacarcin V and M were determined by aserial two-fold agar dilution method. The results shown in Table IVbelow indicate that both albacarcin V and M possess moderategram-positive antibacterial activity with albacarcin V being more potentthan albacarcin M. Little or no gram-negative activity was observed atthe highest dose tested.

                  TABLE IV                                                        ______________________________________                                        Minimum Inhibitory Concentration                                              (mcg/ml)                                                                                          Albacarcin                                                Organism      Strain No.  M        V                                          ______________________________________                                        Streptococcus pneumoniae                                                                    9585        0.4      0.025                                      Streptococcus pyogenes                                                                      9604        1.6      0.05                                       Staphylococcus aureus                                                                       9497        0.8      0.0125                                     Staphylococcus aureus                                                                       9537        3.2      0.0125                                     Staphylococcus aureus                                                                       9606        3.2      0.4                                        Staphylococcus aureus                                                                       20688       1.6      0.1                                        Escherichia coli                                                                            15119       >50      >50                                        Escherichia coli                                                                              20341-1   >50      >50                                        Klebsiella pneumoniae                                                                       15130       >50      >50                                        Proteus mirabilis                                                                           9900        >50      >50                                        Proteus vulgaris                                                                            21559       >50      3.2                                        Serratia marcescens                                                                         20019       >50      >50                                        Enterobacter cloacae                                                                        9659        >50      >50                                        Pseudomonas aeruginosa                                                                        9843A     >50      >50                                        ______________________________________                                    

The antitumor activities of albacarcins V and M were demonstrated by atest against P388 leukemia in mice, the results of which are shown inthe following Table. Details of the methods used in this procedure havebeen described in Cancer Chemother. Rep. 3: 1-87 (Part 3), 1972.

Albacarcins V and M were found to significantly prolong host survival inthe P388 leukemia screening test. Based on comparing both the optimumdoses (maximum T/C value) and minimum effective dose (lowest dose givinga T/C>125), albacarcin V appears to be twice as potent as albacarcin M.

                  TABLE V                                                         ______________________________________                                        Effect of Albacarcin M and V on P-388 Leukemia                                                             Effect                                                     Dose      MST      MST   Survivors                                  Material  mg/kg/inj Days     % T/C Day 5                                      ______________________________________                                        Albacarcin M                                                                            256       16.0     200   6/6                                                  128       14.0     175   6/6                                                  64        12.0     150   6/6                                                  32        12.0     150   6/6                                                  16        9.5      119   6/6                                                   8        9.0      113   6/6                                        Albacarcin V                                                                            256       8.0      100   5/6                                                  128       16.0     200   6/6                                                  64        15.5     194   6/6                                                  32        12.0     150   6/6                                                  16        10.5     131   6/6                                                   8        9.0      113   6/6                                        ______________________________________                                         Host CDF.sub.1 ♀ mice                                                  Treatment 3X, Days 1,4,7                                                      Evaluation MST = median survival time                                         Effect % T/C = (MST treated/MST control) × 100                          Criteria % T/C ≧ 125 considered significant antitumor activity.   

As shown above, albacarcins V and M possess antibacterial activityagainst gram-positive bacteria and are thus useful in the therapeutictreatment of mammals and other animals for infectious diseases caused bysuch bacteria. Additionally, the antibiotics may be used for otherconventional applications of antibacterial agents such as disinfectingmedical and dental equipment.

The marked antitumor activity demonstrated against P388 leukemia in miceindicates that albacarcin V and M are also therapeutically useful ininhibiting the growth of mammalian tumors.

The present invention, therefore, provides a method for therapeuticallytreating an animal host affected by a bacterial infection or by amalignant tumor which comprises administering to said host an effectiveantibacterial or tumor-inhibiting amount of albacarcin V or albacarcin Mor a mixture thereof or pharmaceutical composition thereof.

In another aspect the present invention provides a pharmaceuticalcomposition which comprises an effective antibacterial ortumor-inhibiting amount of albacarcin V or M in combination with aninert pharmaceutically acceptable carrier or diluent. Such compositionmay be made up in any pharmaceutical form appropriate for parenteraladministration.

Preparations according to the invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions oremulsions. They may also be manufactured in the form of sterile solidcompositions whih can be dissolved in sterile water, physiologicalsaline or some other sterile injectable medium immediately before use.

It will be appreciated that the actual preferred amounts of thealbacarcin V or M antibiotic used will vary according to the particularcomposition formulated, the mode of application and the particularsitus, host and disease being treated. Many factors that modify theaction of the drug will be taken into account by those skilled in theart, for example, age, body weight, sex, diet, time of administration,route of administration, rate of excretion, condition of the host, drugcombinations, reaction sensitivities and severity of the disease.Administration can be carried out continuously or periodically withinthe maximum tolerated dose. Optimal application rates for a given set ofconditions can be ascertained by those skilled in the art usingconventional dosage determination tests in view of the above guidelines.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention. Skellysolve B is acommercially available petroleum solvent (Skelly Oil Co.) comprisingisomeric hexanes and having a boiling point of 60°-68° C.

EXAMPLE 1 Fermentation of Albacarcins V and M

A. Shake-flask Fermentation

Streptomyces albaduncus strain C38291 was maintained and transferred intest tubes on agar slants of yeast-malt extract agar. This mediumconsists of 4 g glucose, 4.0 g yeast extract, 10 g malt extract and 20 gagar made up to one liter was distilled water. With each transfer, theagar slant culture was incubated for seven days at 27° C. To prepare aninoculum for the production phase the surface growth from the slantculture was transferred to a 500 ml Erlenmeyer flask containing 100 mlof sterile medium consisting of 30 g glucose, 10 g soy flour, 10 gcottonseed embryo meal and 3 g CaCO₃ made up to one liter with distilledwaer. This vegetative culture was incubated at 27° C. for 48 hours on aGyrotory tier shaker (Model G53, New Brunswick Scientific Co., Inc.) setat 230 rev/min describing a circle with a 5.1 cm diameter. Four ml ofvegetative culture was transferred to a 500 ml Erlenmeyer flaskcontaining 100 ml of sterile production medium consisting of 60 gglucose, 10 g soy flour, 10 g linseed meal, 0.5 g FeSO₄.7H₂ O, 1.0 g NH₄H₂ PO₄ and 10 g CaCO₃ made up to one liter with distilled water. Theproduction culture was incubated at 27° C. on a shaker such as used forthe vegetative culture which was agitated at 230 rev/min. At 196 hoursalbacarcin V yield was 1340 μg/ml and albacarcin M yield was 500 μg/ml.

B. Tank Fermentation

For production of albacarcin V and M in a tank fermentor, 1.9 liters ofvegetative culture was transferred to a stainless steel tank fermentorcontaining 30 liters of production medium previously described. Thetemperature was maintained at 27° C. The air-flow rate was 70liters/min, the back pressure was 1 atm. and the agitation rate was 375rev/min. Polypropylene glycol was used to control foaming. After 134hours the albacarcin V level was 740 μg/ml and albacarcin M level was500 μg/ml.

C. Large Scale Tank Fermentation

For large scale production of albacarcin V and M in a tank fermentor, 1liter of vegetative culture (prepared according to the general procedureof Example 1A) was transferred to a tank fermentor containing sufficientseed medium so as to bring the total volume to 375 liters. Seed mediumconsisted of cerelose (3%), soy flour (1.0%), Pharmamedia (1.0%), CaCO₃(0.3%) and polypropylene glycol (0.05%) in tap water. The seed culturewas incubated at 27° C. The air-flow rate was 100 liters/minute, theback pressure was 5 pounds/sq in and the agitation rate was 155 rev/min.After incubation at 48 hours, 150 liters of the seed culture wastransferred to a 4500 liter tank fermentor containing sufficientproduction medium so as to bring the total volume to 3000 liters.Production medium consisted of cerelose (6.0%), soy flour (1.0%),linseed meal (1.0%), FeSO₄.7H₂ O (0.05%), (NH₄)H₂ PO₄ (0.1%) and CaCO₃(1.0%) in tap water. Incubation was carried out at 27° C. with anair-flow rate of 1400 liters/minute, a back pressure of 15 pounds/sq inand an agitation rate of 155 rev/min. After an incubation period of 156hours, the albacarcin V level was 650 μg/ml and the albacarcin M levelwas 430 μg/ml.

EXAMPLE 2 Isolation of Albacarcins V and M

Step A. Extraction

Raw fermentation whole broth (˜8 l) was transferred to a 80 lpolyethylene tank (48 cm diameter top, 44 cm diameter bottom, 55 cmhigh) equipped with a faucet at the bottom. An equal volume of ethylacetate was added. The mixture was stirred with an air driven stirrer ata good mixing speed for 30 min. Approximately 6 l (2 kg) of Dicalite wasadded and mixed. The mixture was filtered on a Dicalite pad which washeld in a No. 12 Buchner funnel. The filtrate was collected in a 19 lsolution bottle equipped with a vacuum take off. The mat was washed with2 l of ethyl acetate. The filtrate was transferred to a 20 l separatoryfunnel and the phases allowed to separate. Occasionally, an emulsion wasencountered, and this was separated by passing it through a DeLavalGryo-tester centrifuge and collecting the resultant phases. The ethylacetate extract was concentrated to approximately 1 l in a laboratorysize glass circulating evaporator equipped with a continuous feed. Anyprecipitate which formed was collected by filtration. The filtrate wasconcentrated further to approximately 50 ml in vacuo in a rotatoryevaporation. A precipitate was formed by diluting the concentrate with1500 ml of Skellysolve B. The precipitate was collected by filtration toyield approximately 8.3 g of crude solid.

Step B. Liquid Partition of Crude Solid

The crude solid from Step A (8 g) was suspended in 400 ml of methanoland 400 ml of Skellysolve B using an ultrasonic cleaner. The finesuspension was transferred to a 1 l separatory funnel and diluted with44 ml of water. The mixture was shaken and the resultant phases allowedto separate. The aqueous methanol (lower phase) and suspended solidswere transferred to a second separatory funnel. The aqueous methanolphase was extracted three more times with 300 ml aliquots of SkellysolveB. The Skellysolve B had previously been saturated with an equal volumeof 10% water in methanol. The aqueous methanol phase was diluted with 89ml of water and extracted four times with 300 ml portions of carbontetrachloride. The carbon tetrachloride was previously saturated with anequal volume of 25% water in methanol. The aqueous methanol phase wasdiluted with 82 ml of water and extracted for times with 300 ml portionsof chloroform. The chloroform was previously saturated with an equalvolume of 35% water in methanol. All of the suspended solids dissolvedat this point. The chloroform extracts were pooled and evaporated todryness in vacuo in a rotatory evaporator to yield 6.2 g of crudealbacarcin complex.

Step C. Column Chromatography of Crude Complex

A 2.0 cm ID×60 cm Glenco Series 3500 Universal LC column was packed with63 g of Woelm silica gel (70-230 mesh) in methylene chloride. Crudealbacarcin complex (1.1 g) was dissolved in 100 ml of chloroform (2parts) in methanol (1 part). Approximately 7 g of Woelm silica gel wasadded to this solution. The solvent was removed from the mixture invacuo in a rotatory evaporator. The resultant powder was slurried inmethylene chloride and added to the above column. Using a Glencogradient elution apparatus consisting of two chambers of equal diameter,height and volume connected in tandem with Teflon valves, elutioncommenced with a 4 l linear gradient of methylene chloride to 6%absolute ethanol in methylene chloride collecting 20-200 ml fractions.Aliquots (25 μl) of each fraction were spotted on a Whatman LK5DF silicagel tlc plate. The plate was developed with 6% absolute ethanol inmethylene chloride. The desired compounds were detected in fractions9-17 after viewing the plate with 366 nm ultraviolet light. These werepooled and evaporated in vacuo in a rotatory evaporator to yield 889 mgof albacarcin V and M mixture. The chromatography was repeated withfresh crude complex as often as needed to provide albacarcin mixture forsubsequent purifications.

Step D. Preparative HPLC of Albacarcin V/M Mixture

Preparative HPLC was carried out with a Waters Associates Prep LC/System500 instrument charged with two silica gel cartridges (WatersPrepPak-500/Silica). The dry columns were wetted and purged of air withmethylene chloride (approximately 1 liter). The wet columns wereequilibrated with 4 l of 3.5% absolute ethanol in methylene chlorideprior to injection of sample. A flow rate of 350 ml/min was used duringinitial runs. As the columns aged, the flow rate was decreased in 50ml/min increments to 250 ml/min and the chamber pressure graduallyincreased from 23 atmospheres to 28 atmospheres. The effluent wasmonitored with an ISCO model UA-5 absorbance detector at 340 nm-0.5 OD.

Albacarcin V and M mixture (1.08 g) was dissolved in 3 ml ofdimethylsulfoxide. The solution was diluted with 3 ml of methylenechloride and injected immediately into the Prep LC/System 500instrument. Elution commenced using the following program:

1. 1 l eluant to waste,

2. Collect 6-500 ml fractions,

3. Recycle for 8 min 32 sec,

4. Collect 7-500 ml fractions,

5. Recycle for 5 min 43 sec,

6. Collect 500 ml fractions.

The program was adjusted as changes were made in flow rate to achievethe same elution profile and fraction distribution as above. Fractions 5and 6 of the first set, fractions 6 and 7 of the second set, andfractions 6 through 11 of the third set of fractions were pooled andevaporated to approximately 50 ml. A precipitate was created by adding1500 ml of Skellysolve B. The precipitate was collected by filtration toyield 0.54 g albacarcin V. Fractions 1 through 4 of the first set andfractions 14 through 17 of the third set of fractions were pooled andevaporated to about 50 ml. Approximately 1500 ml of Skellysolve B wasadded and a light yellow precipitate resulted. The precipitate wascollected by filtration to yield approximately 300 mg of albacarcin M.The resultant products were assayed by analytical HPLC.

Analytical HPLC of Albacarcins V and M:

The following components were used to construct an analytical HPLCsystem: Waters Associates Model 6000A Solvent Delivery System pump;Varian Varichrom Model VUV-10 uv/vis detector set at 390 nm 0.1 O.D.;Fisher Recorder Series 5000 recorder; Alltech μBondapak C₁₈ (10μ) column(4.6 mm ID×25 cm) with a Whatman CoPell ODS (0.03-0.38 mm) guard column(4.6 mm ID×5 cm). The components were connected with 316 stainless steeltubing (1.6 mm OD-0.12 mm ID). Eluant was pumped at a rate of 2 ml/min.

Using 3 parts water in 2 parts tetrahydrofuran, albacarcin M (k'=1.12)and albacarcin V (k'=1.61), as isolated above, were judged to be 88% and95.5% pure, respectively.

Step E. Final Purification

After numerous preparative runs of additional lots of albacarcin M and Vmixture, the nearly pure albacarcins were rechromatographed bypreparative HPLC, as described above, to yield pure albacarcin M and Vwhich have the physical and spectral properties disclosed earlier in thepresent specification.

We claim:
 1. A process for the production of the antibiotic, albacarcinM, which comprises cultivating Streptomyces albaduncus strain C 38291(ATCC 39151) or an albacarcin M-producing mutant thereof in an aqueousnutrient medium containing assimilable sources of carbon and nitrogenunder submerged aerobic conditions until a substantial amount ofalbacarcin M is produced by said organism in said culture medium andthen recovering the albacarcin M from the culture medium substantiallyfree of co-produced substances.
 2. A process for the production of theantibiotic, albacarcin V, which comprises cultivating Streptomycesalbaduncus strain C 38291 (ATCC 39151) or an albacarcin V-producingmutant thereof in an aqueous nutrient medium containing assimilablesources of carbon and nitrogen under submerged aerobic conditions untila substantial amount of albacarcin V is produced by said organism insaid culture medium and then recovering the albacarcin V from theculture medium substantially free of co-produced substances.
 3. Abiologically pure culture of the microorganism Streptomyces albaduncusATCC 39151, said culture being capable of producing the antibioticsalbacarcins V and M in a recoverable quantity upon cultivation in anaqueous nutrient medium containing assimilable sources of nitrogen andcarbon.